Please note: GeneRead Library Quant Array (product number 180611) and GeneRead Library Quant Kit (product number 180612) will be phased out by the end of 2016. Please use QIAseq Library Quant Assay Kit (product number 333314) instead.

The QIAseq Library Quant System is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

QIAseq Library Quant Array workflow

Each sample DNA library is combined with QIAseq qPCR SYBR® Green Mastermix, aliquoted in technical triplicate across the QIAseq Library Quant Array, and analyzed with qPCR. Raw CT values are copied into the data analysis Excel template, and the software automatically calculates the library concentration or dilution factor.

The QIAseq Library Quant System enables quantification of libraries with concentrations below the detection limit of conventional methods

The QIAseq Library Quant Array's high sensitivity and broad dynamic range enable the quantification of both the NGS-L1 and NGS-L2 libraries [A]. By contrast, Agilent's High Sensitivity DNA Kit (for use with the Agilent 2100 BioAnalyzer) quantified only the NGS-L1 library; with this kit, the NGS-L2 concentration was too low for quantification [B].

Principle of the QIAseq Library Quant System

The serial dilutions of the DNA standard (5 sequential 10-fold dilutions) generate a standard curve. The sample library should fall within the standard curve.

Accurate quantification of library molecules is essential for the efficient use of NGS platforms; however, current spectrophotometric methods only measure total nucleic acid concentrations. The QIAseq Library Quant System, by contrast, uses real-time PCR to quantify only DNA molecules with adaptors at both ends, which are the only amplifiable molecules during emulsion PCR (Ion Torrent platform) or bridge PCR (Illumina platform), and therefore provides highly accurate quantification of amplifiable library molecules. The high sensitivity of real-time PCR allows quantification of libraries at very low concentrations, even below the detection threshold of conventional spectrophotometric methods.

The QIAseq Library Quant System is available in both a tube and an array format, and is optimized with QIAseq qPCR SYBR® Green Mastermixes to provide superior sensitivity and a wide dynamic range. Both formats provide a DNA standard specific to the Illumina or Ion Torrent platform. The array format provides this standard in five predispensed, sequential 10-fold dilutions mixed with a PCR primer assay in triplicate, ensuring that your sample library will fall within the detection range of the array (see figure Principle of QIAseq Library Quant System).

Procedure

The array format workflow (see figure QIAseq Library Quant Array workflow) begins with two tenfold dilutions of the sample library, to ensure that its concentration falls within the range of the serially diluted standards. The samples are mixed with QIAseq qPCR SYBR Green Mastermix and aliquoted in technical triplicate across the array plate. PCR is performed, and raw CT values are exported to the provided data analysis Excel file for automatic calculation of library concentration (Illumina platform) or template dilution factor (Ion Torrent/Proton platform).

The tube format workflow is similar to the array format workflow, with a few small differences. The procedure begins with preparation of five sequential tenfold dilutions of DNA Standard and two tenfold dilutions of the sample library. This ensures that the concentration of the library will fall within the range of the standard dilutions. Next, the diluted DNA standards and sample libraries are mixed with the provided PCR assay and the appropriate QIAseq qPCR SYBR Green Mastermix. PCR is performed, and CT values are exported to the provided data analysis Excel file for automatic calculation of library concentration (Illumina platform) or template dilution factor (Ion Torrent/Proton platform).